DESCRIPTION (PROVIDE BY APPLICANT): Spontaneous intracellular calcium elevations, called transients, occur during embryonic skeletal muscle development. Transients are necessary for myofibrillogenesis (MFG), the process of contractile protein assembly in striated muscle, and the long-term objective of this research is to determine the transduction cascades activated by these signals. Towards this end, the spatiotemporal Ca2. patterns necessary for proper MFG must be examined (Aim 1), since the key parameters have not been defined and may suggest downstream mechanism. It has been hypothesized that transients foment myosin A band assembly by directly activating myosin light chain kinase (MLCK), but this has not been tested (Aim 2). And while transients are necessary for A band assembly, it is unknown if they are also required for establishing Z-ljne periodicity or formation of actin bands (Aim 3). While transients are necessary for MFG in culture, it has not been established that Ca2+ elevations regulate MFG in the embryo (Aim 4). The important temporal patterns of transient production will be established by generating artificial calcium transients using computer-controlled bath application of caffeine. Key temporal and spatial parameters will be examined by localized photolysis of caged calcium. In association with these manipulations. MFG will be analyzed using standard immuriocytochemical methods in fixed cells, or will be actively monitored in living cells. GFP-tagged versions of sarcomeric proteins will be used to monitor myofibril construction in real-time using fluorescence microscopy and high-resolution digital imaging. The degree of MLCK activity correlated with transient production will be assessed with standard biochemical and peptide analytical methods, but a live-imaging possibility will be pursued. In this approach, a fluorescent MLCK sensor which utilizes fluoresence resonant energy transfer (FRET) can determine both the localization and activation of MLCK within live cells. Standard immunocytochemical methods and GFP fusions will also be used to examine Z-line and I band assembly in the presence and absence of normal transients. The calcium-dependence of A and I band interaction necessary for MFG will also be assessed using specific inhibitors of these two assembly processes. For example, a specific pseudosubstrate MLCKi peptide is utilized to inhibit A band assembly and examine the effect on I band formation. Lastly, calcium dynamics and MFG will be simultaneously monitored in the living embryo at high resolution using far-red calcium indicators and sarcomeric GFP fusions, respectively.